1. Field of the Invention
The present invention relates to the fields of molecular biology, virology and immunology. More specifically, the present invention provides replication deficient flaviviruses and discloses its use as vaccine against flavivirus-associated diseases.
2. Description of the Related Art
The Flavivirus genus of the Flaviviridae family contains a variety of important human and animal pathogens that include yellow fever, tick-borne encephalitis, Japanese encephalitis, dengue, West Nile, classical swine fever, bovine viral diarrhea and hepatitis C viruses. In nature, flaviviruses circulate between vertebrate hosts and arthropod vectors mainly represented by a large number of mosquito and tick species. Almost fourty members of this genus, classified into four distinct antigenic complexes, are capable of causing human disease.
The flavivirus genome is a single-stranded RNA of positive polarity of almost 12 kb. It encodes a single polypeptide that is co- and post-translationally processed by cellular and viral proteases into viral structural proteins, C, prM/M, and E, that form infectious viral particles, and the nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5, that form the enzyme complex required for replication of viral genome (Lindenbach and Rice, 2001). The flavivirus genome mimics the structure of cellular messenger RNAs by having a 5′ methylguanylate cap, but differs from the cellular RNA templates by the absence of a 3′-terminal poly(A) sequence.
In flavivirus virions, a single copy of viral genomic RNA is packaged by the C (capsid) protein into nucleocapsid surrounded by lipid envelope with embedded dimers of E and the M protein. The mechanism of interaction between the nucleocapsid and the envelope is not completely understood yet, but it appears to be less specific than, for instance, the alphavirus nucleocapsid-envelope interaction, and the flavivirus virions can be efficiently formed by capsid and envelope proteins derived from the viruses that belong to distant antigenic complexes (Chambers et al., 1999; Lorenz et al., 2002; Monath et al., 2002). Moreover, the presence of nucleocapsid is not an absolute requirement for particles assembly, and virus-like particles formation and release from the cells can be achieved by expression of only prM and E from a wide variety of vectors. These so-called subviral particles (SVPs) contain no RNA or capsid protein (Mason et al., 1991), but have the envelope proteins organized into icoshedral, lipid-containing structure. The prM/E-embedded subviral particles are capable of inducing an efficient immune response that protects animals against following infection with the replication-competent viruses (Konishi and Fujii, 2002; Konishi, Fujii, and Mason, 2001; Konishi et al., 1992; Qiao et al., 2004), DNA (Aberle et al., 1999; Colombage et al., 1998; Davis et al., 2001; Kochel et al., 1997; Kochel et al., 2000; Konishi et al., 2000a; Konishi et al., 2000b; Phillpotts, Venugopal, and Brooks, 1996; Schmaljohn et al., 1997). The lack of nucleocapsid-packaged replication-competent RNA makes application of subviral particles as potential vaccines very advantageous, but requires development of new means for their large-scale production or delivery of the expression constructs. The prM/E-expressing cassettes can be designed on the basis of viral and nonviral vectors. In the case of viral vectors, there is always a concern of either the development or pre-existence of the immune response to the used viral vector. The DNA-based cassettes, encoding these genes under control of efficient RNA polymerase II-based promoters, appear to be preferential. However, their application in clinical practice remains questionable. Therefore, vaccination against flaviviruses is still mainly achieved by using either inactivated or live-attenuated vaccines (INVs and LAVs, respectively).
Recent studies suggested that flavivirus structural proteins are dispensable for the RNA genome replication. They can be either completely or partially deleted and such RNAs (replicons) remain self-replicating and capable of expressing not only the nonstructural, but also remaining structural and/or additional heterologous genes. For example, the flavivirus genomes lacking a functional capsid gene, but having the other structural genes intact were synthesized in vitro and used directly for immunization. Their replication led to the subviral particle production and ultimately induced a protective immune response. Application of the modified flaviviruses that are incapable of developing productive, spreading infection is a new means of designing safe and effective in producing protective immunity vaccines (Aberle et al., 2005; Kofler et al., 2004). However, their application probably requires an improvement of the delivery of the in vitro synthesized RNAs into the cells susceptible for RNA replication. This can be achieved by using the most natural approach, by packaging these defective genomes into infectious particles composed by viral structural proteins.
Despite a great concern for flavivirus-associated diseases and continuing spread of the flaviviruses into the new areas, antiviral therapeutics have not been developed yet for these infections, and a very limited number of approved vaccines have been produced to-date. Inactivated viral vaccines (INV) have been licensed to prevent tick-borne encephalitis (TBEV) and Japanese encephalitis (JEV). However, like other inactivated viral vaccines, these vaccines have limited potency, require multiple vaccinations and are expensive to produce. Despite these drawbacks the Japanese encephalitis and tick-borne encephalitis inactivated viral vaccines have a good safety record, and have not been associated with development of any disease. The only licensed live-attenuated vaccine (LAV) for a flavivirus is the widely utilized yellow fever virus (YFV) 17D strain that was developed by serial passaging of the wt Asibi strain of yellow fever virus in chicken embryo tissues. Although this live-attenuated vaccine is considered very safe and effective, there have been cases of yellow fever and adverse effects detected in vaccines, including a recent case in a US military recruit.
The development of the reverse genetics systems for flaviviruses has opened an opportunity for the designing of new types of live-attenuated vaccine, based on rational attenuation of these viruses. This new class of vaccines includes YFV 17D-based chimeras in which the yellow fever virus prM-E-encoding genome fragment has been replaced with the prM-E-cassette derived from heterologous flaviviruses. Similar chimeric virus-based approach was applied for dengue- and tick-borne encephalitis-based backbones. In most cases, chimeric flaviviruses demonstrate a highly attenuated phenotype, but are capable of eliciting efficient protective immune response and protect against following infection with viruses, whose structural proteins are expressed by the chimeras. Vaccination with these chimeric vaccine candidates is not prevented by pre-existing “vector” immunity, which has interfered with potency of recombinant viral vaccines based on other viral vectors.
Although chimeric flaviviruses appear to provide a reasonably universal approach to producing new vaccines, there are concerns that the chimeras themselves will be pathogenic at least in the immunocompromised individuals, or that pathogenic chimeras may arise since mutations have been detected during the process of propagation of these viruses that will be needed to prepare vaccines.
Thus, prior art is deficient is deficient in a safe, potent and effective type of vaccine that can be used against the Flavivirus genus. The present invention fulfills this long-standing need and desire in the art.